The relative roles of pyruvate kinase and malic enzyme in substrate cycling between pyruvate and oxaloacetate were examined in perfused livers of 24-hour—fasted normal and triiodothyronine (T₃)-treated rats using an inhibitor of malic enzyme (hydroxymalonate). Livers were perfused for 60 minutes in a recirculating system with [3-¹³C]alanine (10 mmol/L, 99% ¹³C-enriched). The combined flux through pyruvate kinase plus malic enzyme relative to pyruvate carboxylase flux was assessed by the ¹³C-enrichment ratio of alanine C2 to glucose C5 in the perfusate, determined with ¹³C and ¹H nuclear magnetic resonance (NMR) spectroscopy. In normal rat livers, the relative carbon flux through pyruvate kinase plus malic enzyme to pyruvate carboxylase was 0.18 ± 0.04, and increased to 0.44 ± 0.08 (P < .05) in the T₃-treated group. After addition of hydroxymalonate, this relative carbon flux was unchanged in normal rat livers, but decreased to 0.15 ± 0.04 (P < .01) in the T₃-treated group, suggesting that the increased carbon flux in T₃-treated livers was caused by increased flux through malic enzyme. Malic enzyme activity increased from 0.36 ± 0.05 U/g liver in normal livers to 2.51 ± 0.50 U/g liver (P < .05) in the T₃-treated group, whereas there was no effect of T₃ treatment on pyruvate kinase activity. We conclude that (1) carbon flux through malic enzyme relative to pyruvate carboxylase flux is minimal in the liver of normal 24-hour—fasted rats, (2) T₃ treatment stimulates substrate cycling between pyruvate and oxaloacetate by increasing carbon flux through malic enzyme, and (3) under hyperthyroid conditions, substrate cycling between pyruvate carboxylase and malic enzyme accounts for a major fraction of the gluconeogenic flux.